Disease states, such as hyperlipidemia, diabetes and cancer, occur when cells allocate too many of their resources towards a metabolic pathway at the cost of other essential pathways. PAS kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice and man, yet little is known about the molecular mechanisms of its function. In this proposal, two recently discovered PAS kinase substrates, Cbf1 and Pbp1, will be further characterized. Yeast PAS kinase was found to inhibit cellular respiration through the phosphorylation and inhibition of Cbf1, while the human Cbf1 homologue (USF1) has been associated with hyperlipidemia in many studies. Therefore, Cbf1/USF1 appears to reduce lipid biogenesis while simultaneously stimulating respiration. This phosphorylation of USF1 may explain many of the pleiotropic effects seen in PAS kinase-deficient mice, particularly the hypermetabolism and resistance to liver triglyceride accumulation. Aim 1 is focused on characterizing the pathways by which Cbf1/USF1 functions to increase respiration at the expense of lipid biogenesis. This includes assaying for USF1 phosphorylation by human PAS kinase (hPASK), characterizing the mitochondrial pathways involved in the upregulation of respiration, and identifying the lipids that are altered in yeast ad mammalian cells. The phosphorylation of yeast Pbp1 by PAS kinase was also recently reported, which inhibits cell growth and proliferation through sequestration of TORC1 at stress granules. This inhibition of TORC1 may explain the neurotoxicity associated with human alleles of the Pbp1 homologue, Ataxin-2. In the second Aim, the role of PAS kinase in Pbp1 and Ataxin-2 regulation will be characterized. In addition, Ataxin-2 will be assessed for its ability to complement yeast Pbp1 function, which may provide a model for studying Ataxin-2 function. The third aim will focus on assaying the effects of PAS kinase on cell growth and proliferation, including cell cycle progression effects. Both Cbf1/USF1 and Pbp1/Ataxin-2 have been reported to affect cell growth and proliferation, positing a role for PAS kinase in cell proliferation contrl. Through this broad phylogenic study, our understanding of the molecular mechanisms that link glucose metabolism with cell growth and proliferation will be expanded. These mechanisms are likely to play key roles in the development of metabolic diseases such as hyperlipidemia, diabetes and cancer.